Intestinal and splenic T cell responses to enteric Listeria monocytogenes infection: distinct repertoires of responding CD8 T lymphocytes.

Listeria monocytogenes is an intracellular bacterium that causes systemic infections after traversing the intestinal mucosa. Clearance of infection and long term protective immunity are mediated by L. monocytogenes-specific CD8 T lymphocytes. In this report, we characterize the murine CD8 T cell response in the lamina propria and intestinal epithelium after enteric L. monocytogenes infection. We find that the frequency of MHC class Ia-restricted, L. monocytogenes-specific T cells is approximately 4- to 5-fold greater in the lamina propria than in the spleen of mice after oral or i.v. infection. Although the kinetics of T cell expansion and contraction are similar in spleen, lamina propria, and intestinal epithelium, high frequencies of Ag-specific T cells are detected only in the lamina propria 1 mo after infection. In contrast to MHC class Ia-restricted T cells, the frequency of H2-M3-restricted, L. monocytogenes-specific T cells is decreased in the intestinal mucosa relative to that found in the spleen. In addition to this disparity, we find that MHC class Ia-restricted CD8 T cells specific for a dominant L. monocytogenes epitope have different TCR V beta repertoires in the spleen and intestinal mucosa of individual mice. These findings indicate that the intestinal mucosa is a depot where L. monocytogenes-specific effector CD8 T cells accumulate during and after infection irrespective of immunization route. Furthermore, our results demonstrate that CD8 T cell populations in these two sites, although overlapping in Ag specificity, are distinct in terms of their repertoire.

Specific resistance of T cells to CD95-induced apoptosis during S phase of the cell cycle.

We have examined the susceptibility of mouse thymocytes and Con A-activated mature T cells to CD95 (Fas; APO-1)-induced apoptosis at different phases of the cell cycle. Signaling through CD95, induced by murine CD95 ligand expressed on fibroblasts, resulted in the preferential apoptosis of T cells in G0-G1 phase of the cell cycle. T cells in S phase were selectively protected. CD95-induced apoptosis was inhibited by exogenous IL-2, which increases the percentage of cells in S phase. The inverse relationship between DNA synthesis and apoptosis in CD95-ligated T cells was not observed during the spontaneous death of T cells in culture or during propriocidal apoptosis due to TCR cross-linking, to which cells in S phase were susceptible. These results show that in T cells there is no distinctively apoptosis-vulnerable phase of the cell cycle; instead, apoptosis can occur at different phases of the cycle depending on the apoptotic stimulus.

Beta2 integrins and ICAM-1 are involved in establishment of the intestinal mucosal T cell compartment.

Development of the mucosal immune system was examined in mice with partial loss of expression of ICAM-1 or CD18. Profound effects on Peyer's patch (PP), lamina propria (LP), and intraepithelial lymphocyte (IEL) T cell populations were observed in mutant mice. Normal expression of CD18 integrins and ICAM-1 was essential for development of the CD8(alpha beta) TCR(alpha beta)LP and IEL compartment and for the generation of normal PP lymphocyte populations. The partial loss of CD8(alpha beta) IEL correlated with the loss of TCR(alpha beta) IEL-mediated lytic activity. The presence of a subset of Thy1+TCR(gamma delta)IEL was also dependent on CD18 integrins and ICAM-1. Both the lytic activity and the expression of CD11c by TCR(gamma delta)IEL were up-regulated in the presence of TCR(alpha beta) T cells. Analysis of bone marrow chimeras demonstrated that a bone marrow-derived ICAM-1+ accessory cell was involved in the generation of some TCR(alpha beta) IEL. These results demonstrated that ICAM-1 and beta2 integrins were required for establishment of a normal intestinal immune system.

Antigen-driven induction of CD11c on intestinal intraepithelial lymphocytes and CD8+ T cells in vivo.

Intraepithelial lymphocytes (IEL) of the intestinal epithelium represent a phenotypically and functionally distinct subpopulation of peripheral T cells. In this study, we report the production of a mAb, designated HL3, which exhibits reactivity with a subset of IEL. In differential screening assays HL3 reacted with 30 to 50% of IEL, but not with T cells of the thymus, spleen, or lymph nodes. Biochemical characterization revealed that the HL3 mAb recognized p150,95 (CD11c/CD18; CR4), a member of the beta 2-integrin family. Fluorescence flow cytometric analyses showed that p150,95 was expressed by TCR-alpha beta or TCR-gamma delta CD4-8+ IEL but not by CD4+8- IEL. Induction of graft-vs-host (GVH) disease resulted in up-regulation of p150,95 expression on donor-derived CD8+ T cells in the intestinal epithelium, as well as in the spleen and lymph nodes. GVH also induced MAC-1 (CD11b) expression on a subset of CD8+ lymph node T cells, but MAC-1 was not up-regulated on CD8+ IEL in this situation. In contrast, activation of identical T cell responders in vitro resulted in weak induction of p150,95 and MAC-1 expression. This result suggested that activation alone was insufficient for p150,95 up-regulation and that additional factors available in vivo were essential in this process. In the intestine, induction of p150,95 required the presence of intestinal flora as IEL from germfree mice lacked p150,95. Interestingly, gamma delta IEL expressing a non-IEL type transgenic TCR were also p150,95-, but exposure to Ag in vivo, but not in vitro, resulted in p150,95 induction. This result indicated that the constitutive expression of p150,95 on IEL is likely due to Ag stimulation via the TCR and not a bystander phenomenon. Overall, the results demonstrated p150,95 to be a hallmark of T cell activation in vivo and an indicator of ongoing antigen-specific T cell activation in the intestinal epithelium.

Regulation of MHC class I synthesis and expression by human neutrophils.

Human peripheral blood neutrophils (PMN) treated with granulocyte-macrophage CSF (GM-CSF) increase the amount of class I 42-kDa H chain and 12-kDa L chain, beta 2-microglobulin (beta 2m), that they synthesize by 2.1- and 2.6-fold, respectively. To determine whether the increase in translation was associated with an increase in levels of class I H chain transcript, RNA blot analysis was performed on PMN that had been cultured in the presence of GM-CSF. Under no conditions were there increased levels of class I H chain transcript when class I heterodimer protein synthesis was increased. In addition, there was neither an increase in the synthesis of H chain mRNA, as measured by transcription assay, nor an alteration in the degradation rates of class I H chain transcript in PMN cultured with GM-CSF. In situ hybridization demonstrated that both the percentage of PMN that expressed class I transcript and the relative amounts of transcript per cell in GM-CSF-cultured PMN were the same as those in control PMN. Although there is increased translation of class I heterodimer in PMN treated with GM-CSF, there is no increase in its expression on the plasma membrane. The maintenance of constant levels of class I on the plasma membrane is dependent on continued protein synthesis and is maintained by release of class I heterodimer and free beta 2m into the medium. Heterodimer is released in the context of plasma membrane-derived vesicles, whereas beta 2m is released as a soluble protein. Maintenance of constant levels of class I heterodimer on the plasma membrane is also regulated by constitutive internalization. Up to 30% of class I molecules bearing 125I-Fab-labeled mAb to class I are internalized over 2 h at 37 degrees C. Therefore, inducible synthesis of class I by PMN is likely a consequence of post-transcriptional regulation, whereas the continued synthesis of class I heterodimer is required for maintenance of its expression. Furthermore, there is no increase in class I expression, in spite of increased synthesis, due to the release of class I heterodimer and beta 2m and the internalization of class I heterodimer from the plasma membrane. Thus, PMN are capable of post-transcriptional regulation of protein synthesis and are able to modulate the expression of plasma membrane proteins by regulated expression, release, and internalization.

Granulocyte-macrophage colony-stimulating factor increases synthesis and expression of CR1 and CR3 by human peripheral blood neutrophils.

Polymorphonuclear leukocytes (PMN) constitutively synthesize various plasma membrane proteins including CR1(3) (CD35), CR3 (or Mac-1) alpha-chain (CD11b) and MHC class I. PMN are also able to up-regulate rapidly the expression of CR1 and CR3 to the plasma membrane in response to agonists such as FMLP. To determine whether constitutive PMN translation was static or up-regulatable, PMN were cultured in the presence or absence of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) for 8 h. CR1, CR3 and class I proteins immunoprecipitated from lysates of 35S-methionine pulse-labeled PMN were resolved by SDS-PAGE, fluorographed and quantified by densitometry. GM-CSF-treated PMN synthesized 4.5-fold more class I protein, 3.7-fold more CR1, 2.4-fold more CD11b and 3.4-fold more CR3 beta-chain (CD18), compared with untreated control cells. Actinomycin D treatment of replicate samples of PMN decreased the amount of these proteins synthesized by each group of PMN from 30 to 90%, implying that continued translation was required for the increases in protein synthesis. Nascent CR and class I proteins were inserted into the plasma membrane of PMN, thereby supplementing the molecules already expressed on the cell surface. In addition to these longer term effects of GM-CSF, we observed its acute up-regulatory effects on PMN. GM-CSF induced a five- to 12-fold increase in the expression of CR1 and CR3 on the PMN cell surface within 30 min. These increases were both dose- and time-dependent with maximum up-regulation occurring at 25 pM and at 30 min. In contrast to the long term biosynthetic events, this rapid up-regulation was not dependent on protein synthesis but was due instead to mobilization of CR from intracellular compartments similar to those up-regulated by FMLP. These results demonstrate that PMN can respond to microenvironmental stimuli such as GM-CSF both by rapidly up-regulating and increasing translation and expression of functionally important plasma membrane proteins.